Isolating traffic-enhancing mutants of drug delivery protein

ABSTRACT

The invention relates to methods for isolating traffic-enhancing mutants of drug delivery proteins. In one embodiment, the invention provides a carrier for delivering a therapeutic agent to an organelle, comprising a polypeptide encoded by a mutant penton base gene. In another embodiment, the invention provides a method of enhancing trafficking to a cell by administering a composition comprising a penton base (PB) protein with one or more mutations that enhance cellular entry.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is filed under 35 U.S.C. § 371 as the U.S.national phase of International Application No. PCT/US2013/053493, filedAug. 2, 2013, which designated the U.S. and claims the benefit ofpriority to U.S. provisional application No. 61/679,306, filed Aug. 3,2013, each of which is hereby incorporated in its entirety including alltables, figures, and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 17, 2016, isnamed 761542000300seqlist.txt and is 39 kilobytes in size.

GOVERNMENT RIGHTS

The invention was made with government support under Grant Nos. CA129822and CA140995 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF INVENTION

The invention provides methods and compositions for cell penetrationfunction to improve drug delivery to specific organelles.

BACKGROUND OF THE INVENTION

Directed evolution has been used to generate pseudotypedadeno-associated virus (AAV) capsids with novel tropism, reducednon-specific delivery, and immune evasion (Kwon and Schaffer, 2008;Maheshri et al., 2006). Biopanning of phage display and whole virallibraries in vitro and in vivo with different selective pressures hasgenerated proteins with desired properties such as improved ligandbinding and uptake, immune interactions, or enzyme activities (Yuan etal., 2005). Thus, this methodology can be a powerful and more effectivealternative to rational mutation for creating new protein variants withimproved features. The most recently developed approach to this processemploys error-prone PCR and staggered extension to create a library ofvariants that can be screened against different cell types to select outcell-specific targeted vectors (Zhao et al., 1998). To date, a processhas not yet been developed to isolate protein variants with improvedintracellular trafficking functions. This invention introduces a newprocedure and new protein molecules with improved cell penetrationfunctions for drug delivery resulting from the procedure.

SUMMARY OF THE INVENTION

Various embodiments include a method of enhancing trafficking to a cell,comprising providing a composition comprising a penton base (PB) proteinwith one or more mutations that enhance cellular entry and administeringan effective dosage of the composition to the cell. In one embodiment,the one or more mutations is 111C and/or 333F. In another embodiment,the composition targets the cytoplasm and/or nucleus of the cell. Inanother embodiment, the composition further comprises a therapeuticdrug. In another embodiment, the cell is a tumor cell. In anotherembodiment, the one or more mutations is SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20.

Other embodiments include a method for producing a drug deliverymolecule that targets an organelle. The method includes the followingsteps: a) obtaining a polynucleotide encoding the penton base (PB) geneand generating mutants of the polynucleotide, b) cloning the mutantpolynucleotide into a phage vector and generating a phage librarycomprising the phage vectors, c) transforming cells with the phagelibrary, d) fractioning the transformed cells and harvesting theorganelle from the transformed cells, e) amplifying the phages from theharvested organelles, f) transforming cells with the amplified phagesfrom the harvested organelle, g) repeating steps (d), (e), (f), and (g),h) titering the phages from the harvested organelles from each round, i)selecting the phages with the highest titer and obtaining the sequencesof the mutant polynucleotide from the phage, and j) producingpolypeptides encoded by the sequences, where the polypeptides are thedrug delivery molecules that target organelles. In another embodiment,the organelle is selecting from the group consisting of mitochondrion,Golgi apparatus, endoplasmic reticulum, nucleus, ribosomes, plasmamembrane and cytosol. In another embodiment, the cells are mammalian ornon-mammalian cells. In another embodiment, the mutants are generatedusing any one or more of PCR-based methods, chemical mutagenesis,ultraviolet-induced mutagenesis or a combination thereof. In anotherembodiment, the drug delivery molecule comprises a targeting domain, anendosomolytic ligand domain and a positively charged domain.

Other embodiments include a carrier for delivering a therapeutic agentto an organelle, comprising a polypeptide encoded by one or more pentonbase (PB) mutations that enhance cellular entry. In another embodiment,the one or more penton base (PB) mutations include 111C and/or 333F. Inanother embodiment, the carrier further comprises a polylysine motif. Inanother embodiment, the carrier further comprises a targeting domain ofheregulin. In another embodiment, the one or more PB mutations comprisesa C-terminal deletion.

Other embodiments include a therapeutic agent comprising a carrier fordelivering a therapeutic agent to an organelle comprising a polypeptideencoded by one or more penton base (PB) mutations that enhance cellularentry and a therapeutic drug. In another embodiment, the therapeuticdrug is a chemotherapeutic agent.

Various other embodiments include a method of producing a carrierwithout proliferative activity, comprising a) obtaining a polynucleotideencoding the receptor binding domain of heregulin (Her) and generatingmutants in the polynucleotide, b) cloning the mutant Her polynucleotidesinto phage vectors and generating a phage library comprising the phagevectors, c) transforming MDA-MB-435 cells with the phage library in thepresence of mitotic inhibitors, d) fractioning the transformed cell andextracting the membrane fraction of the MDA-MB-435 cells, e) harvestingmembrane phages from the membrane fraction, f) transforming the membranephages into MDA-MB-435 cells in the presence of mitotic inhibitors, g)repeating steps (d), (e), and (f), h) monitoring MDA-MB-435 cellproliferation in each round and selecting the membrane phages with thelowest MDA-MB-435 cell proliferation, i) obtaining the sequences of theHer polynucleotide mutants in the selected membrane phages, and j)producing polypeptides encoded by the Her sequences and penton basegene, where the polypeptides are the carrier without proliferativeactivity.

Other embodiments include a carrier for delivering therapeutics to thenucleus, comprising a polypeptide encoded by mutant Her sequences. Inanother embodiment, the carrier further comprises a polypeptide encodingpenton base (PB) protein and a polylysine motif. In another embodiment,the PB protein is a mutant penton base protein.

Other embodiments include a therapeutic agent comprising a carrier and atherapeutic drug.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts, in accordance with an embodiment herein, the biopanningstrategy.

FIG. 2 depicts, in accordance with an embodiment herein, a PCR-basedrandom mutagenesis of the penton base gene. The PCR product (just abovethe 1600 bp band) contains 100 ng DNA, based on densitometry analysis,giving us a total yield of ˜4800 ng. As the initial target DNA was 97ng, we have a yield/initial ratio of ˜50 and a duplication number of˜5.6, which when extrapolated against the standard curve provided in themanufacturer's protocol (Genemorph II, Strategene, La Jolla, Calif.,USA), corresponds to a mutation rate of ˜8/kb.

FIG. 3 depicts, in accordance with an embodiment herein, isolation ofphage displaying penton base variants with enhanced partitioning innuclear compartment. A T7 phage library displaying randomly mutagenizedPB was added to 1×10^6 HeLa cells at 1×10^8 pfu following the conditionsdescribed in the text for cell binding and uptake. Cells were harvestedby trypsinization to remove any surface-bound phage, then fractionatedusing a commercial fractionation kit (Qproteome Cell Compartment Kit;Qiagen Inc., Valencia, Calif., USA). Phage was PEG-precipitatedovernight from each fraction following standard procedures, thenresuspended in TB bacterial media and added to BLT5403 bacteria toamplify the isolated phage. Amplified phage was titered by plaque assay,then re-panned on HeLa using the same titer and conditions as describedearlier. The phage obtained from cytosolic fractions underwent 3 roundsof cytosolic biopanning, whereas that obtained from nuclear fractionsunderwent 2 rounds.

FIG. 4 depicts, in accordance with an embodiment herein, the alignmentof trafficking variants isolated from biopanning.

FIG. 5 depicts, in accordance with an embodiment herein, full-lengthmutant, 111C, exhibits enhanced trafficking to cytoplasm and nucleus.FIG. 5(A) depicts protein was precipitated from each fraction by acetoneprecipitation and pellets were resuspended in 40 uL desalting buffer,followed by analysis via SDS-PAGE and Western blotting. FIG. 5(B)depicts analysis of band densities using Image J show an increase of111C in both cytosolic and nuclear fractions compared to the wild-typeprotein. Trafficking and Cell fractionation assay: Adherent HeLa cellsgrowing in flasks were detached with 5 mL 1×PBS+2 mM EDTA at 37 Cincubation with agitation for 50 min and transferred to 15 mL conicaltubes. The density of the cells were measured, and cells weredistributed into separate tubes at 5×106 cells per tube. Cells werewashed with 1×PBS+Ca2++Mg2+ three times to remove the EDTA, and cellpellets were resuspended in 0.7 ml Buffer A (20 mM HEPES, pH 7.4; 2 mMMgCl2; 3% BSA in DMEM). Wild-type (WT) or mutant (111C, 333F) pentonbase protein (450 ug) was added to separate cell aliquots and mixtureswere incubated at 4C for 2 hrs with agitation to promote receptorbinding, followed by incubation at 37C for 2 h with agitation to promoteinternalization of receptor-bound protein. Control treatments receivedno protein (NP). Cells were then collected and processed for subcellularfractionation using the Qproteome Cell Compartment assay kit (Qiagen).

FIG. 6 depicts, in accordance with an embodiment herein, PB mutants,111C and 333F, exhibit enhanced nuclear entry compared to wild-type PB.FIG. 6(A) depicts intracellular trafficking and immunocytochemistry.Procedure followed the established protocols detailed in Gene Therapy(2006) 13, 821-836. Anti-penton base antibody (Ad5 antibody) was used at1:500 dilution. Alexa-Fluor 488 Goat Anti-Rabbit was used at 1:400dilution (2nd antibody). Phalloidin was used at 1:100 dilution, and DAPIused at 300 nM. Green, wild-type (WT) or mutant (111C, 333F) PB; Red,actin; Blue, nucleus. FIG. 6(B) depicts quantification of proteintrafficking Sixteen cells from each treatment represented in the leftpanel was selected for quantification. The counts were based on thehistogram of each image in Adobe Photoshop. Bars represent green pixelcounts within the 80-255 window in the green channel.

FIG. 7 depicts, in accordance with an embodiment herein, an updatedAlignment of PB Mutants. New peptide lengths are based on amino acidsequences translated from nucleic acid sequences of mutant clones.Sequences are provided in FIGS. 8-17 herein.

FIG. 8 depicts, in accordance with an embodiment herein, nucleic Acidand peptide sequence of mutated PB from Fraction 111 Clone A (111A).

FIG. 9 depicts, in accordance with an embodiment herein, nucleic acidand predicted amino acid sequence of mutated PB from Fraction 111 CloneC (111C).

FIG. 10 depicts, in accordance with an embodiment herein, nucleic acidand peptide sequence of mutated PB from Fraction 111 Clone G (111G).

FIG. 11 depicts, in accordance with an embodiment herein, nucleic acidand peptide sequence of mutated PB from Fraction 331 Clone E (331E).

FIG. 12 depicts, in accordance with an embodiment herein, nucleic acidand peptide sequence of mutated PB from Fraction 331 Clone I (331I).

FIG. 13 depicts, in accordance with an embodiment herein, nucleic acidand peptide sequence of mutated PB from Fraction 331 Clone J (331J).

FIG. 14 depicts, in accordance with an embodiment herein, nucleic acidand peptide sequence of mutated PB from Fraction 333 Clone A (333A).

FIG. 15 depicts, in accordance with an embodiment herein, nucleic acidand peptide sequence of mutated PB from Fraction 333 Clone D (333D).

FIG. 16 depicts, in accordance with an embodiment herein, nucleic acidand peptide sequence of mutated PB from Fraction 333 Clone E (333E).

FIG. 17 depicts, in accordance with an embodiment herein, nucleic acidand peptide sequence of mutated PB from Franction 333, clones F, G, andH (333F, 333G, 333H).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the abbreviation “PB” means penton base.

A variety of reagents have been developed to deliver therapeutic genesand drugs to diseased cells, and include liposomes, synthetic polymers,peptides, proteins, viruses, and viral nanoparticles (Medina-Kauwe,2006; MedinaKauwe et al., 2005). Typically, the particles formed bythese reagents require modifications to facilitate delivery of gene ordrug payloads. Such modifications include appending targeting ligands orantibodies, membrane penetrating agents, and/or intracellular targeting(such as nuclear targeting) agents. These modifications are usuallyintroduced through rational design and thus each new variant generatedby such modification requires empirical testing. This can be timeconsuming and labor intensive, and runs the risk of yielding suboptimalactivity.

Past and present attempts to improve cell membrane penetration and/orintracellular trafficking have used rational design to conjugate cellpenetration or intracellular targeting peptides to drug carriers. Suchan approach requires empirical testing of each molecule.

Numerous types of cell penetration peptides have been tested forenhanced gene and drug delivery, and include AntP, TAT, GALA, honey beemelittin, and similar peptides (Medina-Kauwe, 2006; Medina-Kauwe et al.,2005). Likewise, nuclear targeting activity has been added to genedelivery agents via appendage of different poly-basic domains such asthe SV40 NLS, HMG-1, protamine, and similar peptides or proteins(MedinaKauwe, 2006; Medina-Kauwe et al., 2005). While each of thesepeptides possess the capacity to penetrate cell membranes or targetintracellular compartments, including the nucleus, their activities arealtered when covalently coupled to another molecule or expressed as afusion protein to another molecule. Moreover, empirical testing of eachdifferent variant of these peptides is time consuming and laborintensive. Therefore, while rational design and empirical testing is theexisting solution to this problem, this approach, has its limitations.

The invention described herein circumvents the time and effort requiredfor rational design and empirical testing of cellpenetration/intracellular trafficking proteins/peptides by usingselective pressure to isolate protein mutants that have acquiredimproved cell penetration, intracellular trafficking, and/or subcellulartargeting. The protein mutants derived from this process have uniqueadvantages over the parent proteins or existing gene/drug deliveryproteins currently in use because of the improved features acquiredthrough artificial evolution/selective pressure. Finally, the specificproteins isolated by the process described below will provide anadvantage over existing cell penetrating peptides because of theirimproved membrane lysis and trafficking features. Therefore, they can beused to augment gene and drug delivery, and thus enhance therapeuticefficacy of particles used in nanomedicine.

The invention provides a method for producing a drug delivery moleculethat targets an organelle. The method includes the steps of (a)obtaining a polynucleotide encoding the penton base gene and generatingmutants of the polynucleotide; (b) cloning the mutant polynucleotideinto a phage vector and generating a phage library comprising the phagevectors; (c) transforming cells with the phage library; (d) fractioningthe transformed cells and harvesting the organell from the transformedcells; (e) amplifying the phages from the harvested organelles; (f)transforming cells with the amplified phages from the harvestedorganelle; (g) repeating steps (d), (e), (f), and (g); (h) titering thephages from the harvested organelles from each round; (i) selecting thephages with the highest titer and obtaining the sequences of the mutantpolynucleotide from the phage; and (j) producing polypeptides encoded bythe sequences, wherein the polypeptides are the drug delivery moleculesthat target organelles.

In some embodiments, the organelle is selecting from the groupconsisting of mitochondrion, Golgi apparatus, endoplasmic reticulum,nucleus, ribosomes, plasma membrane and cytosol. The cells may bemammalian or non-mammalian cells. Mutants may be generated using anyrandom mutagenesis methods or targeted mutagenesis methods. Examples ofmethods that may be used to generate mutants include but are not limitedto any one or more of PCR-based methods, chemical mutagenesis,ultraviolet-induced mutagenesis or a combination thereof. Mutations inthe penton base gene may be any one or more of insertions, deletions,substitutions or a combination thereof.

In an embodiment of the invention, the drug delivery molecule comprisesa targeting domain, an endosomolytic ligand domain and a positivelycharged domain.

The invention also provides a carrier for delivering a therapeutic agentto an organelle, comprising a polypeptide encoded by a mutant pentonbase gene. The mutation in the penton base gene may be isolated by a)obtaining a polynucleotide encoding the penton base gene and generatingmutants of the polynucleotide; (b) cloning the mutant polynucleotideinto a phage vector and generating a phage library comprising the phagevectors; (c) transforming cells with the phage library; (d) fractioningthe transformed cells and harvesting the organell from the transformedcells; (e) amplifying the phages from the harvested organelles; (f)transforming cells with the amplified phages from the harvestedorganelle; (g) repeating steps (d), (e), (f), and (g); (h) titering thephages from the harvested organelles from each round; (i) selecting thephages with the highest titer and obtaining the sequences of the mutantpolynucleotide from the phage; and (j) producing polypeptides encoded bythe sequences, wherein the polypeptides are the drug delivery moleculesthat target organelles. The carrier further comprises a polylysine motifand a targeting domain, for example the targeting domain of heregulin.

The invention further provides a therapeutic agent comprising thecarrier described above and a therapeutic drug. A therapeutic drug maybe any drug that, for example, treats, inhibits, prevents, mitigates theeffects of, reduce the severity of, reduce the likelihood of developing,slow the progression of and/or cure, a disease. Diseases targeted by thetherapeutic agents include but are not limited to carcinomas, sarcomas,lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed onvarious immune cells, and antigens expressed on cells associated withvarious hematologic diseases, autoimmune diseases, and/or inflammatorydiseases. Therapeutic agents may be a chemotherapeutic agent.

The invention also provides a method of producing a carrier withoutproliferative activity. The method comprises (a) obtaining apolynucleotide encoding the receptor binding domain of heregulin (Her)and generating mutants in the polynucleotide; (b) cloning the mutant Herpolynucleotides into phage vectors and generating a phage librarycomprising the phage vectors; (c) transforming MDA-MB-435 cells with thephage library in the presence of mitotic inhibitors; (for exampletaxol); (d) fractioning the transformed cell and extracting the membranefraction of the MDA-MB-435 cells; (e) harvesting membrane phages fromthe membrane fraction; (f) transforming the membrane phages intoMDA-MB-435 cells in the presence of mitotic inhibitors; (g) repeatingsteps (d), (e), and (f); (h) monitoring MDA-MB-435 cell proliferation ineach round and selecting the membrane phages with the lowest MDA-MB-435cell proliferation; (i) obtaining the sequences of the Herpolynucleotide mutants in the selected membrane phages; and (j)producing polypeptides encoded by the Her sequences and the penton basegene, wherein the polypeptides are the carrier without proliferativeactivity. Mutations in the Her gene may be any one or more ofinsertions, deletions, substitutions or a combination thereof.

The invention further provides a carrier for delivering therapeutics tothe nucleus, comprising a polypeptide encoded by the mutant Hersequences wherein the mutant Her is obtained according to the methoddescribed above. The carrier further comprises a polypeptide encodingpenton base protein and a polylysine motif. The penton base protein maybe a mutant protein.

EXAMPLES Example 1 Isolation of Traffic-Enhancing Mutants

Previously, a recombinant adenovirus penton base protein was developedto target and deliver a variety of therapeutic molecules to tumor cellsin vitro and in vivo (Agadjanian et al., 2012; Agadjanian et al., 2009;Agadjanian et al., 2006; Medina-Kauwe et al., 2001a; Medina-Kauwe etal., 2001b; Rentsendorj et al., 2008). Currently, the penton baserecombinant protein (the same one that comprises the ‘PB’ domain ofHerPBK10, used to target therapeutics to HER2+ tumor cells; (Agadjanianet al., 2012; Agadjanian et al., 2009; Agadjanian et al., 2006;MedinaKauwe et al., 2001b; Rentsendorj et al., 2008) proceeds throughmultiple cell entry routes after cell binding, some but not all of whichsupport membrane penetration and entry into the cytosol (Rentsendorj etal., 2006). To improve upon this function and enhance the delivery andpenetration of therapeutics into cell targets, a directed evolutionapproach was used to isolate penton base variants with enhanced cellpenetration activity by using nuclear accumulation as a readout, asendosomal escape enables entry into the nucleus (Rentsendorj et al.,2006).

Procedures:

This two-step process involves: 1. Creation of a mutant library throughrandom mutagenesis, and 2. Introduction of a selective pressure toisolate variants with improved function, depending on the screeningprocess. Here, isolation of phage that survive entry into the cytosoland/or nucleus will serve as the selective pressure, which is expectedto yield variants with enhanced cell penetration activity (Summarized inFIG. 1). Accordingly, the inventors have randomly mutagenized the pentonbase gene and generated a library of mutants cloned into a K. T7 phagevector. Based on previously established directed evolution studies(Cherry et al., 1999; Shafikhani et al., 1997; Wan et al., 1998; You andArnold, 1996), the inventors aimed for a mutation frequency of 1-4 aminoacid changes (or 2-7 nucleotide changes) per gene. Based on the productyield using a specialized error-prone polymerase chain reaction (PCR)method (GeneMorphII Random Mutagenesis Kit; Stratagene, La Jolla,Calif., USA), the inventors achieved an estimated mutation frequency of˜8 nucleotides/kb or 13.6 nucleotides per penton base gene (FIG. 2). Theinventors inserted this product into a T7-Select phage vector andpackaged recombinant phage to produce an amplified library titer of5×10¹⁰ pfu/mL.

The library was panned onto HeLa cells (which express integrin receptorsfor binding and uptake of the PB protein). Phage were incubated on thecells at 4° C. for 1 h to promote receptor binding but not uptake, thencells were washed and incubated for 2 h at 37° C. to promotedinternalization. After uptake, harvested cells underwent fractionationto isolate cytosolic and nuclear fractions. Phage amplified from eachfraction then underwent repeated biopanning, and correspondingsequential fractions were extracted from cell harvests (i.e. nuclearphage isolated from round 1 were amplified and added back to cells,followed by repeat isolation of nuclear fractions to re-obtain nuclearphage). After either two or three rounds of biopanning, phage isolatedfrom each repeat fraction was titered to determine the relativeenrichment of nuclear/cytosolic phage from the mutagenized librarycompared to phage displaying wild-type penton base.

Results:

The non-mutagenized parent phage, T7-PB, yielded a nuclear/cytosolicphage titer ratio of less than 1, indicating that the proportion ofphage arriving at the nucleus by 2 h was less than the proportion ofphage that remained in the cytoplasm (FIG. 3). In contrast, after 2rounds of biopanning and isolation of nuclear phage (3-3a), a shift wasobserved toward higher nuclear accumulation compared to cytoplasmicretention, with a significant increase compared to T7-PB (P=0.05) (FIG.3). Even phage isolated from 3 rounds of cytosolic fraction panning(1-1-1) showed a relative, though not highly significant (P=0.07),increase in nuclear partitioning compared to T7-PB (FIG. 3).

After three rounds of biopanning and isolation of cytosolic and nuclearmutants, the inventors sequenced clones picked randomly from eachenriched population and found that the majority of clones isolated fromboth cytosolic and nuclear fractions encoded carboxy-[C-] terminaltruncated protein (FIG. 4). The 287LDV and 340RGD integrin bindingmotifs located near the middle of the wild-type penton base (wt PB)linear sequence were not retained in most of the truncated clones. Oneof the truncated mutants contains the LDV but not RGD motif, whereas theremaining truncations lack both LDV and RGD motifs. The full-lengthclones isolated from the biopanning retain both LDV and RGD motifs butalso contain several point mutations that introduce potentialfunction-altering amino acid changes (FIG. 4). Among these are aLeu60Trp replacement in cytosolic fraction clone 111C; and Lys375Glu,Val449Met, and Pro469Ser amino acid changes in nuclear fraction clone333F. To test the ability of each isolated variant to impart enhancedcytosolic and/or nuclear penetration, the intracellular trafficking ofeach will be compared to the parent protein by immunofluorescence andconfocal microscopy, and confirmed by subcellular fractionation.Specifically, as the full-length mutants (111C and 333F) and mutant 331Jretain the integrin binding motifs, these variants are tested incomparison to wt PB, as they are predicted to enter cells via integrinbinding and uptake. Meanwhile, as the remaining truncated variants lackany receptor-binding motifs, these will be inserted into HerPBK10 toreplace the PB domain, and tested in comparison to parental HerPBK10,which enters cells via human epidermal growth factor receptor (HER)binding and uptake.

Example 2 Isolation of Receptor-Binding and Endocytosis Mutants withBlunted Signaling

Rationale:

The tumor-targeted cell penetration protein, HerPBK10, is specificallydirected to the human epidermal growth factor receptor (HER) viainclusion of the receptor binding domain of heregulin, designated hereas the ‘Her’ domain of HerPBK10 (Medina-Kauwe et al., 2001b). However,ligation of heregulin receptors can induce signaling that may result intumor cell proliferation, differentiation, and in some cases, apoptosis,depending on several factors including receptor heterodimer ratio,ligand subtype, cell type, and presence of certain intracellularmolecules (Aguilar et al., 1999; Lewis et al., 1996; Weinstein et al.,1998). The possibility of inducing adverse effects such as tumorprogression leads to examining whether the selective pressure introducedby mitotic inhibitors can select receptor binding and endocytosismutants that lack proliferative signaling. The inventors proposedtesting this approach by generating a phage library displaying Hervariants and screen the library for internalizing Her species lackingproliferative signaling by isolating internalized phage from quiescentcells, and re-panning on non-proliferating human breast cancer cells.

Procedure:

A library of Her sequences containing different mutations distributedacross the coding sequence will be produced by error-prone PCR andstaggered extension, which entails repeated cycles of denaturation andbrief annealing/extension after initial priming of template sequences(Zhao et al., 1998). The resulting library will be inserted into theappropriate vector arms for transfer into T7Select bacteriophage (whichis developed to display whole proteins), and recombinant phage producedfollowing manufacturer's instructions (Novagen, Gibbstown, N.J., USA).Based on biopanning of evolved AAV capsids, an initial titer of 10′12phage will be added to MDA-MB-435 cells maintained in media containingmitotic inhibitor such as taxol, and at about 30-45 min later (the timerequired for binding and internalization; Medina-Kauwe et al., 2000) thecells will be harvested by trypsin/EDTA (to remove non-internalizedphage) and membrane-extracted to isolate the vesicle fraction of crudevirus (Qproteome Plasma Membrane Protein Kit, Qiagen Inc., Valencia,Calif., USA), which can then be isolated by CsCl banding. Three to fourrounds of selection (i.e. adding isolated virus to fresh cells andrepeating membrane extraction) will be performed and isolated virus willbe characterized in the following ways. First, fresh cells receivingenriched virus will be fixed and processed for immunofluorescence usingan anti-phage antibody (Sigma-Aldrich, St. Louis, Mo., USA) to confirmthat the isolated phage still internalize. Separate cells treated inparallel will be assessed for proliferation rate in comparison to mockand untreated cells, by metabolic assay. Second, the Her sequence fromisolated phage will be excised and inserted into a bacterial expressionvector for recombinant protein production (Medina-Kauwe et al., 2001a),then mutant Her tested for internalization and proliferative activity inMDA-MB-435 human breast cancer cells as described earlier, in comparisonto parental Her as well as mock and untreated cells. The mutant cloneswill be sequenced to identify mutated regions, and inserted back intothe HerPBK10 expression cassette, replacing parental ‘Her’.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (for example, “such as”) provided withrespect to certain embodiments herein is intended merely to betterilluminate the application and does not pose a limitation on the scopeof the application otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the application.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

In closing, it is to be understood that the embodiments of theapplication disclosed herein are illustrative of the principles of theembodiments of the application. Other modifications that can be employedcan be within the scope of the application. Thus, by way of example, butnot of limitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

What is claimed is:
 1. A method of enhancing nuclear subcellularlocalization of a carrier polypeptide in a cell, comprisingadministering the carrier polypeptide to the cell, the carrierpolypeptide comprising a penton base polypeptide, wherein: the pentonbase polypeptide comprises one or more mutations selected from the groupconsisting of Met1Thr, Ser14Tyr, Val18Ala, Pro31Thr, Leu60Trp, Asn74Tyr,Leu170Met, Lys375Glu, Val449Met, and Pro469Ser, the amino acid numberingaccording to SEQ ID NO:
 20. 2. The method of claim 1, wherein the pentonbase polypeptide comprises: (i) residues 37-322 of SEQ ID NO: 4; or (ii)SEQ ID NO:
 20. 3. The method of claim 1, wherein the carrier polypeptideis complexed with a therapeutic drug or a gene.
 4. The method of claim1, wherein the cell is a tumor cell.
 5. The method of claim 1, whereinthe penton base polypeptide comprises residues 37-322 of SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO:
 20. 6. Acarrier polypeptide, comprising a penton base polypeptide, wherein: thepenton base polypeptide comprises one or more mutations selected fromthe group consisting of Met1Thr, Ser14Tyr, Val18Ala, Pro31Thr, Leu60Trp,Asn74Tyr, Leu170Met, Lys375Glu, Val449Met, and Pro469Ser, the amino acidnumbering according to SEQ ID NO:
 20. 7. The carrier polypeptide ofclaim 6, further comprising a positively-charged domain.
 8. The carrierpolypeptide of claim 6, further comprising a cell-targeting domain. 9.The carrier polypeptide of claim 6, wherein the carrier polypeptide iscomplexed with a therapeutic drug or a gene.
 10. The carrier polypeptideof claim 6, further comprising a polypeptide encoded by a mutant Hersequence produced by: (a) mutating a receptor binding domain of aheregulin (Her) polynucleotide to form a mutant Her polynucleotide; (b)cloning the mutant Her polynucleotide into phage vectors and generatinga phage library comprising the phage vectors; (c) transforming breastcancer cells with the phage library in the presence of mitoticinhibitors; (d) fractioning the transformed cell and extracting themembrane fraction of the breast cancer cells; (e) harvesting membranephages from the membrane fraction; (f) repeating steps (c), (d), and(e); and (g) obtaining the sequences of the Her polynucleotide mutantsin the selected membrane phages.
 11. The carrier polypeptide of claim10, further comprising a polylysine motif.
 12. The carrier polypeptideof claim 10, wherein the carrier polypeptide is complexed to atherapeutic drug or a gene.
 13. The carrier polypeptide of claim 6,wherein the penton base polypeptide comprises Met1Thr and Leu60Trpmutations, the amino acid numbering according to SEQ ID NO:
 20. 14. Thecarrier polypeptide of claim 6, wherein the penton base polypeptidecomprise Lys375Glu, Val449Met, and Pro469Ser mutations, the amino acidnumbering according to SEQ ID NO:
 20. 15. The carrier polypeptide ofclaim 6, wherein the penton base polypeptide comprises: (i) residues37-322 of SEQ ID NO: 4; or (ii) SEQ ID NO:
 20. 16. The carrierpolypeptide of claim 7, wherein the positively-charged domain is apolylysine motif.
 17. The carrier polypeptide of claim 8, wherein thecell-targeting domain is heregulin or a mutant thereof.
 18. The carrierpolypeptide of claim 16, further comprising a cell-targeting domain. 19.The carrier polypeptide of claim 18, wherein the penton base polypeptidecomprises: (i) residues 37-322 of SEQ ID NO: 4; or (ii) SEQ ID NO: 20.20. The carrier polypeptide of claim 18, wherein the cell-targetingdomain is heregulin or a mutant thereof.
 21. The carrier polypeptide ofclaim 20, wherein the penton base polypeptide comprises: (i) residues37-322 of SEQ ID NO: 4; or (ii) SEQ ID NO:
 20. 22. The carrierpolypeptide of claim 8, wherein the cell-targeting domain targets acancer cell.
 23. The carrier polypeptide of claim 22, wherein the pentonbase polypeptide comprises: (i) residues 37-322 of SEQ ID NO: 4; or (ii)SEQ ID NO:
 20. 24. The carrier polypeptide of claim 8, wherein thecell-targeting domain targets a mammalian cell.
 25. The carrierpolypeptide of claim 8, wherein the cell-targeting domain targets adiseased cell.
 26. The carrier of claim 6, wherein the penton basepolypeptide comprises residues 37-322 of SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 20.